U.S. patent application number 10/742335 was filed with the patent office on 2004-09-30 for tissue-specific transporter inhibitor.
Invention is credited to Miyamoto, Ken-ichi, Oya, Toru, Sai, Yoshimichi, Tamai, Ikumi, Tsuji, Akira, Yui, Noubuhiko.
Application Number | 20040191211 10/742335 |
Document ID | / |
Family ID | 19027864 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040191211 |
Kind Code |
A1 |
Tsuji, Akira ; et
al. |
September 30, 2004 |
Tissue-specific transporter inhibitor
Abstract
The present invention is to provide a tissue-specific
transporter inhibitor which is not absorbed through the digestive
tract and can prevent deterioration in the QOL of a patient caused
by diet therapy, and a therapeutic drug for tissue dysfunction
diseases and a therapeutic drug for suppressing the progress of
chronic renal failure containing the inhibitor as an active
ingredient. A tissue-specific transporter inhibitor which is not
absorbed through the digestive tract is constructed by introducing
a dipeptide which is a ligand of an oligopeptide transporter 1 into
a supramolecular structure polyrotaxane wherein its structurally
modified active residue is expected to be excellent in the
interaction with a transmembrane transporter.
Inventors: |
Tsuji, Akira; (Kanazawa-shi,
JP) ; Tamai, Ikumi; (Kanazawa-shi, JP) ; Sai,
Yoshimichi; (Kanazawa-shi, JP) ; Yui, Noubuhiko;
(Nomi-gun, JP) ; Oya, Toru; (Ishikawa-gun, JP)
; Miyamoto, Ken-ichi; (Tokushima-shi, JP) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
19027864 |
Appl. No.: |
10/742335 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10742335 |
Dec 19, 2003 |
|
|
|
PCT/JP02/06104 |
Jun 19, 2002 |
|
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Current U.S.
Class: |
424/78.19 |
Current CPC
Class: |
B82Y 5/00 20130101; A61K
47/6951 20170801; A61K 31/724 20130101; A61P 1/00 20180101; A61K
38/05 20130101; A61K 31/765 20130101; A61P 43/00 20180101; A61K
47/6949 20170801; A61P 13/12 20180101 |
Class at
Publication: |
424/078.19 |
International
Class: |
A61K 031/785 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2001 |
JP |
2001-188843 |
Claims
1. A tissue-specific transporter function inhibitor which has both
a ligand structure recognized by a tissue-specific transporter and
a polymeric molecular structure incapable of passing through a
membrane tissue.
2. The tissue-specific transporter function inhibitor according to
claim 1, wherein the polymeric molecular structure incapable of
passing through a membrane tissue is a supramolecular
structure.
3. The tissue-specific transporter function inhibitor according to
claim 2, wherein the supramolecular structure is a rotaxane
compound in which a number of circular molecules are penetrated by
linear molecules, and both ends of the linear molecules are capped
by bulky substituents.
4. The tissue-specific transporter function inhibitor according to
claim 3, wherein the circular molecules are cyclodextrins.
5. The tissue-specific transporter function inhibitor according to
claim 3, wherein the linear molecules are polyethyleneglycols.
6. The tissue-specific transporter function inhibitor according to
claim 3, wherein the bulky substituents are
N-benzyloxycarbonyl-L-phenylalanine- s.
7. The tissue-specific transporter function inhibitor according to
claim 1, wherein the polymeric molecular structure incapable of
passing through a membrane tissue is an .alpha.-cyclodextrin
structure.
8. The tissue-specific transporter function inhibitor according to
claim 1, wherein the ligand recognized by a tissue-specific
transporter is an organic anionic substance, an organic cationic
substance, or a peptidergic substance.
9. The tissue-specific transporter function inhibitor according to
claim 1, wherein the tissue-specific transporter is a small
intestine-specific transporter.
10. The tissue-specific transporter function inhibitor according to
claim 9, wherein the small intestine-specific transporter is an
oligopeptide transporter 1 (PEPT 1).
11. The tissue-specific transporter function inhibitor according to
claim 10, wherein a peptidergic substance recognized by the
oligopeptide transporter 1 (PEPT 1) is valyl-lysine (Val-Lys).
12. A therapeutic drug for tissue dysfunction diseases which
contains the tissue-specific transporter function inhibitor
according to claim 1 as an active ingredient.
13. A therapeutic drug for suppressing the progress of chronic
renal failure which contains the tissue-specific transporter
function inhibitor according to claim 1 as an active ingredient,
wherein the inhibitor is a protein absorption inhibitor.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application PCT/JP02/06104 filed Jun. 19, 2002 and published as WO
03/000285 on Jan. 3, 2003, which claims priority from Japanese
Application 2001-188843, filed Jun. 21, 2001.
[0002] Each of the applications and patents cited in this text,
including each of the foregoing cited applications, as well as each
document or reference cited in each of the applications and patents
(including during the prosecution of each issued patent;
"application cited documents"), and each of the PCT and foreign
applications or patents corresponding to and/or claiming priority
from any of these applications and patents, and each of the
documents cited or referenced in each of the application cited
documents, are hereby expressly incorporated herein by reference.
More generally, various documents or references are cited in this
text, either in a Reference List before the claims or in the text
itself; and, each of the documents or references ("herein cited
documents") and all of the documents cited in this text (also
"herein cited documents"), as well as each document or reference
cited in each of the herein cited documents (including any
manufacturer's specifications, instructions, etc. for products
mentioned herein and in any document incorporated herein by
reference), is hereby expressly incorporated herein by reference.
There is no admission that any of the various documents cited in
this text are prior art as to the present invention. Also,
teachings of herein cited documents and documents cited in herein
cited documents and more generally in all documents incorporated
herein by reference can be employed in the practice and utilities
of the present invention.
FIELD OF THE INVENTION
[0003] The present invention relates to a tissue-specific
transporter function inhibitor which has both a ligand structure
recognized by a tissue-specific transporter and a polymeric
molecular structure incapable of passing through a membrane tissue,
and a therapeutic drug for tissue dysfunction diseases or a
therapeutic drug for suppressing the progress of chronic renal
failure containing the tissue-specific transporter function
inhibitor as an active ingredient, and the like.
BACKGROUND
[0004] Nowadays, the number of dialysis patients are increasing,
and it is presumed that the number of such patients will be
enormous when taking into account the number of diabetic patients
who will be in need of dialysis in the future, and the medical
expenses for dialysis is estimated to be well over 1 trillion yen.
Considering these situations, preventive medicine that prevents the
onset of renal diseases and conservative treatments that prevent
the progress of renal failure into dialysis are regarded important.
Effective treatments of renal disorders in patients of chronic
renal failure have not been established yet, treatments including
low protein diet therapy and the administration of antihypertensive
drugs such as an ACE inhibitor have been conducted so far (Am. J.
Cardiol. 59, 66A-71A, 1987; Am. J. Kidney. 20, 443-57, 1992; BMJ
304, 216-20, 1992; Ann. Intern. Med. 124, 627-32, 1996). The
above-mentioned low protein diet therapy is thought to be effective
means to suppress the progress of chronic renal failure and is
widely conducted currently. However, since dietary restriction
contains problems of quality of life (QOL) and compliance of
patients, a new therapeutic strategy, for example, suppression of
oral protein absorption, is needed. Recently, as a new strategy for
the treatment of hyperlipemia, it is reported that biosynthesis of
cholesterol is suppressed by inhibiting a bile acid transporter
present in the small intestine, and this report draws attention (J.
Pharmacol. Exp. Ther. 293, 315-20 2000). Likewise, it is expected
that the absorption of proteins through the digestive tract can be
suppressed by a specific inhibitor.
[0005] The present inventors have reported that proteins taken are
digested in the digestive tract to amino acids and oligopeptides
and absorbed through the small intestine, and that the absorption
is conducted by a specific transporter present in the brush border
membrane of a small intestine epithelial cell (Pharm. Res. 13,
963-77, 1996). The digested amino acids mentioned above are
transported by multiple transporters, however, the oligopeptides
are transported by an oligopeptide transporter such as PEPT 1, and
absorbed dipeptide- or tripeptide-specifically (J. Biol. Chem. 270,
6456-63, 1995). As to the absorption of digestive products of
proteins in the small intestine, it is known that more peptides are
absorbed than amino acids (Gastroenterology 113, 332-40, 1997).
Taken together, it is considered that a PEPT 1 inhibitor is capable
of suppressing the absorption of proteins in the diet, and is
useful for the patients whose QOL is deteriorated due to diet
therapy.
[0006] Since 1994, PEPT 1 genes have been cloned from small
intestines of rabbit, human and rat (J. Biol. Chem. 270, 6456-63,
1995; Nature 368, 563-6, 1994; J. Pharma. Exp. Ther. 275, 1631-7,
1995; Biochim. Biophys. Acta, 1305, 34-8, 1996), and studies for
transportation via PEPT 1 have been rapidly developed. The
above-mentioned PEPT 1 gene derived from rat small intestine has
been cloned for the first time by the present inventors (Biochim.
Biophys. Acta, 1305, 34-8, 1996), and revealed to locate in the
brush border membrane side of the small intestine epithelial cell
by immunohistochemical technique (FEBS Lett. 392, 25-9, 1996). In
addition, it is reported that PEPT 1 recognizes and transports
compounds such as valacyclovir, an antiviral drug, that does not
have a peptide bond in its molecules, as well as compounds having
peptide-like structures, for example, .beta.-lactam antibiotics
(Pharm. Res. 13, 963-77, 1996; Biochem. Biophys. Res. Commun. 250,
246-51, 1998; J. Clin. Invest. 101, 2761-7, 1998; J. Biol. Chem.
273, 20-2, 1998). As mentioned above, PEPT 1 shows wide range of
substrate recognition property, however, its molecular recognition
property remains unknown and it is thought that the substrate
recognition of PEPT 1 involves not only the recognition of partial
structure but also whole molecule. Meanwhile, PEPT 2, which is
cloned from the kidney (Biochim. Biophys. Acta, 1235, 461-6, 1995;
Biochim. Biophys. Acta, 1280, 173-7, 1996; Proc. Natl. Acad. Sci.
USA 93, 284-9, 1996), locates in the brush border membrane side of
the epithelial cell in the proximal convoluted tubule of the
kidney, and has a substrate recognition property similar to that of
PEPT 1, and serves to reabsorbing oligopeptides and peptide-like
compounds. The above-mentioned PEPT 1 is known to express in the
kidney though it does not contribute very much (Am. J. Physiol.
276, F658-65, 1999). However, PEPT 2 has never been observed to
express in the small intestine.
[0007] In human, it is reported that bioavailability (BA) of
cefadroxil (CDX), a .beta.-lactam antibiotic and a substrate of
PEPT 1, is decreased by coadministration of cephalexin (CEX), a
.beta.-lactam antibiotic similarly recognized by PEPT 1 (Eur. J.
Clin. Pharmacol. 41, 179-83, 1991). A mechanism in which AUC (Area
Under the plasma concentration Curve) as an index of
bioavailability is decreased by CEX includes both the absorption of
CDX in the small intestine and the inhibition of reabsorption of
CDX in the kidney. The reabsorption through the kidney is conducted
mainly via an oligopeptide transporter (PEPT 2), and both compounds
are known to be substrates for PEPT 2 (Biochim. Biophys. Acta,
1235, 461-6, 1995). Therefore, it is explicable that the decrease
of BA of CDX caused by CEX means that CDX transportation via PEPT 1
and PEPT 2 is inhibited by CEX. Though the effect of the inhibition
of PEPT 2 present in the kidney on a living organism is unknown, it
seems preferable to limit to a direct inhibition of absorption via
PEPT 1 from the viewpoint of diet therapy for chronic renal
failure. However, since PEPT 1 and PEPT 2 show very similar
substrate recognition properties, it has been presumed to be
difficult to develop an inhibitor which specifically recognizes
PEPT 1.
[0008] The number of dialysis patients due to renal failure are
increasing, and it is presumed that the number of such patients
will be enormous when taking into account the number of diabetic
patients who will be in need of dialysis, and the medical expenses
for dialysis is estimated to be well over 1 trillion yen in the
future. Under these circumstances, preventive medicine that
prevents the onset of renal diseases and conservative treatments
that prevent the progress of renal failure into dialysis are
important. The object of the present invention is to provide a
tissue-specific transporter inhibitor which is not absorbed through
the digestive tract and can prevent deterioration in the QOL of
patients caused by diet therapy, and a therapeutic drug for tissue
dysfunction diseases and a therapeutic drug for suppressing the
progress of chronic renal failure containing the inhibitor as an
active ingredient.
[0009] The present inventors have considered that it is effective
to use a PEPT 1 inhibitor which is not absorbed through the
digestive tract and is able to avoid recognizing PEPT 2 in order to
attain the above-mentioned object, and that PEPT 1 can be
selectively inhibited by designing a polymer compound having PEPT 1
recognition property because polymer compounds are not absorbed
through the digestive tract in general. Therefore, the present
inventors have focused on a supramolecular structure polyrotaxane
(PRX) wherein its structurally modified active residue is expected
to be excellent in the interaction with a transmembrane
transporter, and constructed a compound wherein a dipeptide
(Val-Lys) which is a ligand of the PEPT 1 mentioned above is
introduced into a supramolecular structure PRX. As a result of
intensive study, it has been found that the above-mentioned
compound can suppress the absorption of proteins and the progress
of chronic renal failure which needs limitation of protein uptake,
and thus the present invention has been completed.
BRIEF DESCRIPTION OF THE INVENTION
[0010] The present invention relates to a tissue-specific
transporter function inhibitor which has both a ligand structure
recognized by a tissue-specific transporter and a polymeric
molecular structure incapable of passing through a membrane tissue
(paragraph 1), the tissue-specific transporter function inhibitor
according to paragraph 1, wherein the polymeric molecular structure
incapable of passing through a membrane tissue is a supramolecular
structure (paragraph 2), the tissue-specific transporter function
inhibitor according to paragraph 2, wherein the supramolecular
structure is a rotaxane compound in which a number of circular
molecules are penetrated by linear molecules, and both ends of the
linear molecules are capped by bulky substituents (paragraph 3),
the tissue-specific transporter function inhibitor according to
paragraph 3, wherein the circular molecules are cyclodextrins
(paragraph 4), the tissue-specific transporter function inhibitor
according to paragraph 3 or 4, wherein the linear molecules are
polyethyleneglycols (paragraph 5), the tissue-specific transporter
function inhibitor according to any one of paragraphs 3 to 5,
wherein the bulky substituents are
N-benzyloxycarbonyl-L-phenylalanines (paragraph 6), the
tissue-specific transporter function inhibitor according to
paragraph 1, wherein the polymeric molecular structure incapable of
passing through a membrane tissue is an .alpha.-cyclodextrin
structure (paragraph 7), the tissue-specific transporter function
inhibitor according to any one of paragraphs 1 to 7, wherein the
ligand recognized by a tissue-specific transporter is an organic
anionic substance, an organic cationic substance, or a peptidergic
substance (paragraph 8), the tissue-specific transporter function
inhibitor according to any one of paragraphs 1 to 8, wherein the
tissue-specific transporter is a small intestine-specific
transporter (paragraph 9), the tissue-specific transporter function
inhibitor according to paragraph 9, wherein the small
intestine-specific transporter is an oligopeptide transporter 1
(PEPT 1) (paragraph 10), and the tissue-specific transporter
function inhibitor according to paragraph 10, wherein a peptidergic
substance recognized by the oligopeptide transporter 1 (PEPT 1) is
valyl-lysine (Val-Lys) (paragraph 11).
[0011] The present invention also relates to a therapeutic drug for
tissue dysfunction diseases which contains the tissue-specific
transporter function inhibitor according to any one of paragraphs 1
to 11 as an active ingredient (paragraph 12), and a therapeutic
drug for suppressing the progress of chronic renal failure which
contains the tissue-specific transporter function inhibitor
according to any one of paragraphs 1 to 11 as an active ingredient,
wherein the inhibitor is a protein absorption inhibitor (paragraph
13).
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1 is a schema showing the synthetic procedure of
polyrotaxane.
[0013] FIG. 2 is a schema showing the synthetic procedure of
valyl-lysine derivative recognized by a peptide transporter.
[0014] FIG. 3 is a schema showing the synthetic procedure of
Val-Lys-polyrotaxane conjugate which is the tissue-specific
transporter inhibitor of the present invention.
[0015] FIG. 4 is a graph showing the inhibitory effect of
Val-Lys-polyrotaxanes on [.sup.3H] Gly-Sar uptake by HeLa-hPEPT 1
cells.
[0016] FIG. 5 is a graph showing the effect of preadministration of
Val-Lys-polyrotaxanes or Val-Lys-.alpha.-cyclodextrins on [.sup.3H]
Gly-Sar uptake by HeLa-hPEPT 1 cells.
[0017] FIG. 6 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without cephalexin to rats.
[0018] FIG. 7 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without Val-Lys-polyrotaxane (No. 2) to rats.
[0019] FIG. 8 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without Val-Lys-polyrotaxane (No. 7) to rats.
[0020] FIG. 9 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
CDX, and cephalexin or Val-Lys-polyrotaxane (No. 2) to rats.
[0021] FIG. 10 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without Val-Lys-polyrotaxane (No. 7) to rats.
[0022] FIG. 11 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without Val-Lys-polyrotaxane (No. 7) to rats.
[0023] FIG. 12 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after administration with
or without Val-Lys-polyrotaxane (No. 7) to rats.
[0024] FIG. 13 is a graph showing the result of time course change
of cefadroxil concentration in the plasma after instant intravenous
injection of CDX and simultaneous oral administration with
Val-Lys-polyrotaxane (No. 7) to rats.
DETAILED DESCRIPTION OF THE INVENTION
[0025] As a tissue-specific transporter function inhibitor
according to the present invention, any substance can be used as
long as it has a ligand structure recognized by a tissue-specific
transporter and a polymeric molecular structure incapable of
passing through a membrane tissue, and inhibits the function of the
above-mentioned tissue-specific transporter, however, those that
have physiologically stable structures are preferable. Examples of
the tissue include; small intestine, kidney, brain, liver,
placenta, pancreas, lung, stomach, ovary, testis, spleen, large
intestine, skeletal muscle, airway, bone marrow, prostate gland,
heart, uterus, spinal cord, adrenal gland, thyroid gland, etc., and
specific examples of the transporters which specifically express in
such tissue include, but not limited to, transporters shown in
Tables 1 to 3.
1TABLE 1 Accession No. Reference Type Name (GenBank, NCBI)
(Journal, vol, pages, year) Tissue Distribution Substrate to be
Transported Organic OAT1 AB004559 J. Biol. Chem., 272, 18526-18529,
Kidney, (brain) .beta.-lactam antibiotics (Penicilline G, anion
1997 Cephaloridine), antiviral drugs transporter (azidothymidine,
acyclovir), nonsteroidal antiinflammatory drugs (salicylate,
acetylsalicylate, indomethacin), ACE inhibitor (captopril),
anticancer drug (methotrexate), metabolites (p- aminohippuric acid,
uric acid), ochratoxin A, cAMP, cGMP OAT2 NM_006672 FEBS Lett.,
429, 179-182, Liver, kidney salicylate, acetylsalicylate, 1998
p-aminohippuric acid, prostaglandin E2, etc. OAT3 AB017446 J. Biol.
Chem., 274, 13675-13680, Kidney, liver, brain p-aminohippuric acid,
ochratoxin A, 1999 estron sulfate conjugate, cimetidine, etc. OAT4
J. Biol. Chem., 275, 4507-4512, Placenta, kidney sulfic acid
conjugates of 2000 various pharmaceuticals Organic OATP-A U21943
Biochem. Biophys. Res. Kidney, brain anion Commun., 273, 251-260,
2000 transporter OATP-B AB026256 Biochem. Biophys. Res. Pancreas,
liver, lung, intestinal Estron sulfate conjugate, Commun., 273,
251-260, 2000 tract, ovary, testis, spleen prostaglandin E2 OATP-C/
AB026257 Biochem. Biophys. Res. Liver Taurocholic acid,
pravastatin, LST1/ Commun., 273, 251-260, 2000
dehydroepiandrosterone sulfate OATP-2 conjugate, estradiol
glucuronate conjugate, prostaglandin E2, thromboxane B2,
leukotoriene B2, etc. OATP-D AB031050 Biochem. Biophys. Res. Most
of normal tissues and Estron sulfate conjugate, Commun., 273,
251-260, 2000 cancer cells prostaglandin E2 OATP-E AB031051
Biochem. Biophys. Res. Most of normal tissues and Estron sulfate
conjugate, Commun., 273, 251-260, 2000 cancer cells prostaglandin
E2 PGT NM_005630 Science, 268, 866-869, 1995 Pancreas, lung,
intestinal tract, Prostaglandin (PGE2, ovary PGF2u, D2),
thromboxane ovary B2, etc. oatp1 AF148218 Proc. Natl. Acad. Sci.
USA, 91, Liver, kidney, large intestine, Organic acid (sulfate
conjugate such 133-137, 1994 brain, lung, skeletal muscle as
cholate, taurocholate, BSP, estron sulfate, digitoxin, ouabain,
enalapril, temocaprilat, pravastatin, estron; glucuronate conjugate
such as estradiol; leucotrien C4, ouabain, BQ-123, etc.). oatp2
U88036 Proc. Nat.l. Acad. Sci. USA, Liver, kidney, brain sulfate
conjugate 94, 10346-10350, 1997 such as bile acid, pravastatin,
estron; glucuronate conjugate such as estradiol; thyroid hormone,
ouabain, digoxin, BQ-123, leuci-enkephalin, etc. oatp3 U95001 J.
Biol. Chem., 273, 22395-22401, Liver, brain 1998 OAT-K1 D79981 J.
Biol. Chem., 271, 20719-20725, Kidney 1996 Npt1 X71355 Genomics,
18, 255-359, 1993 Kidney, liver p-aminohippuric acid, .beta.-lactam
antibiotics (Penicilline G, faropenem, etc.), forcarnet, mevalonic
acid
[0026]
2TABLE 2 Accession No. Reference Type Name (GenBank, NCBI)
(Journal, vol, pages, year) Tissue Distribution Substrate to be
Transported Monocarboxylic MCT1 D63834 Biochem. Biophys. Res.
Normal tissue of almost all Lactic acid, .beta.-hydroxy acid
transporter Commun., 217, 370-375, 1995 over the body butyrate,
acetic acid, propionic acid AE2 Benzoic acid, nicotinic acid,
propionic acid, butyric acid, valproic acid Organic cation OCT1
X78855 Nature, 372, 549-552, 1994 Liver, kidney, intestinal Cholin,
dopamine, adrenalin, transporter epithelial cell (blood vessel
tetraethyl ammonium, N- side) methylnicotinamide, cimetidine,
amantadine. OCT2 D83044 Biochem. Biophys. Res. Kidney, brain
Commun., 224, 500-507, 1996 OCT3 AF055286 J. Biol. Chem., 273,
15971-15979, Kidney, brain, intestinal tract 1998 Organic cation
OCTN1 AB007448 FEBS Lett., 419, 107-111, Kidney, airway, bone
marrow, Tetraethyl ammonium, transporter AB016257 1998 skeletal
muscle, prostate quinidine, pyrilamine, gland, lung, pancreas,
verapamil, carnitine placenta, heart, uterus, spleen, spinal cord,
many cancer cells OCTN2 AB015050 J. Biol. Chem., 273, 20378-20382,
Kidney, skeletal muscle, Carnitine, acetylcarnitine, AB015800 1998
placenta, heart, small intestine, pyrilamine, verapamil Nature
Genet. 21, 91-94 prostate gland, adrenal gland, (1999) airway,
thyroid, many cancer cells OCTN3 NM_019723 J. Biol. Chem., in press
(Sep. 28, 2000) Peptide PEPT1 U13173 J. Biol. Chem., 270,
6456-6463, Small intestine, kidney, cancer Dipeptide, tripeptide,
transporter 1995 cell .beta.-lactam antibiotics (cyclacillin,
Biochim. Biophys. Acta, 1305, cephadroxil, cephalexin, 34-38, 1996
cephradine, ceftibuten, etc.), ACE inhibitor (captopril),
anticancer drug (bestatin), antiviral drug (valacyclovir) PEPT2
D63149 Biochim, Biophys, Acta, 1240, Kidney Cefadroxil 1-4,
1995
[0027]
3TABLE 3 Accession No. Reference Type Name (GenBank, NCBI)
(Journal, vol, pages, year) Tissue Distribution Substrate to be
Transported Amino LAT1 AB015432 J. Biol. Chem., 273, 23629-23632,
Neutral amino acid (leucine), acid 1998 amino acid analogues (T3,
T4, L- transporter dopa, gabapentin, merpharan) LAT2 AF171669 J.
Biol. Chem., 274, 19745-19751, Normal tissue of almost all 1999
over the body BAT1 AB029559 J. Biol. Chem., 274, 28845-28848, 1999
Xc- AB022345 J. Biol. Chem., 274, 11455-11458, 1999 y + LAT1
AJ130718 Nature Genet, 21, 293-296, 1999 y + LAT2 NM_012244 EMBO
J., 18, 49-57, 1999 asc1 J. Biol. Chem., 275, 9690-9698, 2000 MDR1
M62425 Cell, 47, 381-389, 1986 Anticancer drugs (daunorubicin,
doxorubicine, etoposide, vinblastine, vincristine, mitomycin C,
paclitaxel) Other pharmaceuticals (digoxin, progesterone, morphine,
rifampicin, diltiazem, nifedipine, erythromycin) MRP1 AJ277881
Science, 258, 1650-1654, Various cancers, kidney, liver, 1992
spleen, adrenal gland, lung, heart, skeletal muscle MRP2 AF261713
Science, 271, 1126-1128, 1996 Liver Glutathione, various
conjugates, MRP3 AF009670 methotrexate, pravastatin, temocaprilat,
new quinolon antimicrobial
[0028] As the above-mentioned polymeric molecular structure
incapable of passing through a membrane tissue, any structure can
be used as long as it is a polymeric structure incapable of or
having difficulty in passing through a membrane tissue in a living
organism, for example, a membrane tissue in small intestine,
kidney, brain, liver, placenta, pancreas, lung, stomach, ovary,
testis, spleen, large intestine, skeletal muscle, airway, bone
marrow, prostate gland, heart, uterus, spinal cord, adrenal gland,
thyroid gland, etc. The specific examples include a supramolecular
structure such as a polyrotaxane compound in which a number of
circular molecules are penetrated by linear molecules, and both
ends of the linear molecules are capped by bulky substituents, and
a derivative or a clathrate structure containing
.alpha.-cyclodextrin. The specific examples of the circular
molecules mentioned above include but not particularly limited to
molecules such as cyclodextrin, .alpha.-, .beta.-, or
.gamma.-cyclodextrin, crown ether, and cyclofructan. As the linear
molecules, molecules such as polyethyleneglycol, polypropylene
glycol, or copolymer of polyethyleneglycol and polypropylene
glycol, polyamino acid, polysaccharides, etc. are exemplified,
however, polyethyleneglycol and the like, to which a bulky
substituent can be introduced, is preferable. The bulky substituent
is not particularly limited as long as it can prevent desorption of
the circular molecules mentioned above, and the specific examples
include but not particularly limited to an oligopeptide comprising
a unit or units of any one of N-benzyloxycarbonyl-L-phenylalanine,
alanine, valine, leucine, isoleucine, methionine, proline,
phenylalanine, tryptophan, aspartic acid, glutamic acid, glycine,
serine, threonine, tyrosine, cysteine, lysine, arginine, histidine,
or derivatives thereof.
[0029] The specific examples of the ligand recognized by a
tissue-specific transporter in the present invention include an
organic anionic substance, an organic cationic substance, a
peptidergic substance and a substance having an amino group. For
example, the specific examples of the ligand recognized by an
oligopeptide transporter 1 (PEPT 1), a transporter that
specifically expresses in the small intestine, include but not
limited to oligopeptides such as a dipeptide and a tripeptide,
derivatives thereof whose constitutive amino acid residues are
modified, .beta.-lactam antibiotics such as cefadroxil and
ceftibuten, ACE inhibitors such as captopril, bestatin which is an
anticancer drug, and valacyclovir which is an antiviral drug.
[0030] As a therapeutic drug for tissue dysfunction diseases
provided by the present invention, a drug that contains the
tissue-specific transporter function inhibitor as an active
ingredient, and as a therapeutic drug for suppressing the progress
of chronic renal failure provided by the present invention, a drug
that contains the tissue-specific transporter function inhibitor
that suppresses protein absorption as an active ingredient are
exemplified respectively. It is preferable for the therapeutic
drugs to have shapes capable of being administered orally,
intravenously, intraperitoneally, intranasaly, intracutaneously,
subcutaneously, intramuscularly, or in other such manners. It is
possible to conveniently determine the effective amount of the
drugs to be administered in consideration of the types and
compositions of the drugs, its administration route, age and body
weight of patients, etc., and it is preferable to administer the
effective amount of the drugs one to a few times a day. Further, in
the case of oral administration, the drugs are usually administered
in a form of a drug prepared by mixing with carriers for
formulation. As the carriers for drug formulation, substances which
are conventionally used in the drug formulation field, and does not
react with the tissue-specific transporter function inhibitor
according to the present invention are used. The oral
administration of the drugs can be conducted at each meal, or
before each meal.
[0031] In addition, specific examples of dosage forms include
tablets, capsules, granules, powders, syrups, suspensions,
suppositories, ointments, creams, gels, transdermal preparations,
respiratory tonics, injectable solutions. These drugs are prepared
according to conventional methods, and liquid drugs, in particular,
can be prepared also in a form that can be dissolved or suspended
in water or other suitable media before use. Tablets and granules
may be coated by known methods. The injectable solutions are
prepared by dissolving the peptide-modified polymers of the present
invention into water, however, if necessary, instead of water,
saline or glucose solution may be used for dissolution, and buffers
or preservatives may be added. These drugs may contain other
therapeutically valuable components.
[0032] It is also possible to blend the tissue-specific transporter
function inhibitor of the present invention, as a food material for
ameliorating the symptoms of tissue dysfunction diseases or chronic
renal failure, into foods and to take such foods as functional
foods. The examples of such foods are: bread and confectionery
including baked goods such as puddings, cookies, bread, cakes,
jellies, rice crackers, Japanese sweets such as "yokan" (a sweet
jelly made from bean jam), frozen desserts, chewing gums; noodles
such as wheat noodles and buck wheat noodles; fish paste products
such as steamed fish paste, fish ham, fish sausages; various
beverages such as yogurt, yogurt drinks, juice, milk, soy milk,
alcoholic drinks, coffee, tea, green tea, oolong tea, isotonic
drinks; seasonings such as miso, soy sauce, dressings, mayonnaise,
sweeteners; various delicatessen such as tofu, devil's tongue,
"tsukudani" (fish boiled on soy sauce), jiao-zi, croquettes, salad,
etc.
EXAMPLES
[0033] Hereinafter, preferred examples of the present invention are
described, however, the present invention is not limited to these
examples.
Example 1
[0034] [Synthesis of Polyrotaxane (PRX); see FIG. 1]
[0035] 1-1 (Preparation of Pseudo-polyrotaxane Comprised of
Polyethyleneglycol and .alpha.-cyclodextrin)
[0036] An aqueous solution (9.14 g/85 ml) of polyethyleneglycol
(PEG-BA, Mn=4000) whose both ends were aminated was dropped into
.alpha.-cyclodextrin (.alpha.-CD) saturated aqueous solution (100
g/600 ml), which was being sonicated during the dropping. The
resultant solution was sonicated and stirred for about one hour,
and left overnight. Then, centrifugation was performed, and
precipitate was collected and dried under reduced pressure at
60.degree. C. Thus, 78.13 g of pseudo-polyrotaxane, a linear
molecule wherein polyethyleneglycol (PEG) was penetrating into
cyclodextrin was prepared.
[0037] 1-2 (Preparation of an End-cap Agent)
[0038] In order to introduce N-benzyloxycarbonyl-L-phenylalanine
(Z-L-Phe, Z represents a benzyloxycarbonyl group) as a bulky
substituent that prevents the desorption of .alpha.-CD, a carboxyl
group in Z-L-Phe was activated. 38.46 g (0.33 mol) of
N-hydroxysuccinimide (HOSu) and 100 g (0.33 mol) of Z-L-Phe were
dissolved in 850 ml of dioxane. Next, 68.90 g (0.33 mol) of
ice-cooled N,N'-dicyclohexylcarbodiimide (DCC) was added and the
resultant solution was stirred for about one hour, and
subsequently, left overnight in a refrigerator. After removing the
precipitate formed, its supernatant was concentrated under reduced
pressure and the obtained concentrated solution was re-precipitated
in diethyl ether. The precipitate formed was dried under reduced
pressure at ambient temperature and collected, and re-crystallized
with dichloromethane and petroleum ether. Then, the sample was
filtrated and dried under reduced pressure, and 105.84 g (0.26 mol)
of succinimide ester of Z-L-Phe (Z-L-Phe-OSu), a white needle
crystal, was obtained.
[0039] 1-3 (Synthesis of Polyrotaxane Using Z-L-Phe-OSu)
[0040] 24.3 g (0.68 mmol) of pseudo-polyrotaxane obtaind in Example
1-1, and 28.8 g (72 mmol) of Z-L-Phe-OSu obtaind in Example 1-2
were added to 30 ml of dimethyl sulfoxide, and the resultant
solution was stirred for about 4.5 days in heterogeneous condition.
In the procedure, the molar ratio of Z-L-Phe-OSu (--OSu) to a
terminal amino group of pseudo-polyrotaxane (--NH.sub.2) was 50:1.
After the reaction, the above solution was poured into a large
amount of ether, and the resultant white precipitate was dried
under reduced pressure and collected. In order to remove
Z-L-Phe-OSu, .alpha.-CD, and PEG-BA that were not reacted, the
precipitate was washed three times each with acetone and water.
Finally the obtained sample was dried under reduced pressure at
60.degree. C., and 13 g of polyrotaxane (white powder), wherein
both ends of pseudo-polyrotaxane were capped by bulky substituents,
was obtained. The structural analysis of the synthesized
polyrotaxane was performed by .sup.1HNMR spectral measurement
(Varian; 300 MHz FT-NMR).
Example 2
[0041] [Synthesis of a Dipeptide Recognized by a Peptide
Transporter]
[0042] A valyl-lysine (Val-Lys: VK) derivative, one of the
dipeptide analogues recognized by a peptide transporter, was
synthesized according to the method described by Abe et al.
(Bioconjugate Chem. 10, 24-31, 1999) (see FIG. 2).
[0043] 2-1 (Synthesis of Boc-Val-Lys(Cbz)-Ot-Bu)
[0044] Tertiary butyloxycarbonyl(Boc)-Val (2.17 g, 10 mmol),
.epsilon.-benzyloxycarbonyl lysine-tert-butyl ester hydrochloride
[Lys-(Cbz)-Ot-Bu-HCl (3.37 g, 10 mmol)], 1-hydroxybenzotriazole
(HOBt) (4.13 g, 20 mmol), and dimethylaminopyridine (DMAP) (1.93 g,
10 mmol) were dissolved in 80 ml of N,N-dimethylformamide (DMF)
respectively, and the resultant solution was stirred for 30 minutes
at 0.degree. C. Subsequently, water-soluble carbodiimide
hydrochloride (WSC.HCl) (1.93 g, 10 mmol) was added and the
resultant solution was stirred for about two hours at 0.degree. C.,
then stirred for about four hours at room temperature, and diluted
with ethyl acetate. The solution diluted with ethyl acetate was
sequentially washed with 0.6 M of citric acid aqueous solution (100
ml), water (100 ml), saturated sodium bicarbonate aqueous solution
(100 ml), water (100 ml), 10% of saline (100 ml). As the result,
the obtained oil layer was dried with sodium sulfate and
concentrated under reduced pressure, then purified by column
chromatography (SiO.sub.2, chloroform:methanol=75:1). Elution peak
was determined by thin-layer chromatography, and the first obtained
fraction was concentrated and dried under reduced pressure, and
Boc-Val-Lys(Cbz)-Ot-Bu in the form of noncrystalline white powder
was obtained (3.6 g, yield: 66%).
[0045] 2-2 (Synthesis of Boc-Val-Lys-Ot-Bu-HCl)
[0046] The Cbz group of Boc-Val-Lys(Cbz)-Ot-Bu obtained in Example
2-1 was deprotected by the catalytic reduction method. In the
presence of H.sub.2 gas, Boc-Val-Lys(Cbz)-Ot-Bu was dissolved in
150 ml of acetic acid, then palladium carbon (300 mg) was added and
the resultant solution was stirred for three days. The palladium
carbon was removed by filtration, the solution was concentrated
under reduced pressure, and the concentrated solution was subjected
to ion exchange chromatography. Diaion WA-30 (HCl form) was used as
an ion exchanger, and methanol-water (10:1) was used as a solvent
for development. After the solution was concentrated under reduced
pressure, azeotropic procedure was conducted with toluene, then
white solid of Boc-Val-Lys-Ot-Bu-HCl (2.2 g, yield: 51%) was
obtained.
Example 3
[0047] [Synthesis of Val-Lys-polyrotaxane Conjugate; See FIG.
3]
[0048] 3-1 (Activation of a Hydroxyl Group in Polyrotaxane by
N,N-carbonyldiimidazole)
[0049] 200 mg of polyrotaxane (--OH: 3 mmol) obtained in Example 1
was dissolved in 10 ml of dimethyl sulfoxide (DMSO) under nitrogen
atmosphere. After the polyrotaxane was completely dissolved, 1000
mg (6.2 mmol) of N,N-carbonyldiimidazole (CDI) was added to the
solution, and the resultant solution was also stirred. Three hours
later, CDI that was not reacted was removed by re-precipitation in
ether, and 374 mg of CDI-activated polyrotaxane (CDI-PRX) was
obtained. The activation ratio of the CDI-PRX mentioned above was
30%, when introduction into all hydroxyl groups was regarded as
100%.
[0050] 3-2 (Introduction of Val-Lys into Polyrotaxane)
[0051] 200 mg of the CDI-PRX mentioned above (--OH: 1.8 mmol) was
dissolved in 2 ml of DMSO under nitrogen atmosphere, and 1300 mg
(3.3 mmol) of Boc-Val-Lys-Ot-Bu.HCl obtained in Example 2 and 700
.mu.l (3.5 mmol) of N,N-diisopropylethylamine (DIPEA) were added
and the resultant solution was stirred for 24 hours. Subsequently,
a hydroxyethylcarbamoyl (HEC) group was introduced into
polyrotaxane, and 2 ml (33 mmol) of aminoethanol (AMEt) was added
in order to improve water solubility, and then the resultant
solution was stirred for 24 hours. After stirring, dialysis was
conducted in water using dialytic membrane with molecular weight
cut off 1000. After the dialysis was completed, polyrotaxane (PRX)
into which Boc-Val-Lys-Ot-Bu was introduced was collected by
freeze-drying. In ice-cooled condition, the collected substance was
dissolved in a mixed solution of 7 ml of dichloromethane (DCM) and
3 ml of trifluoroacetate (TFA), and the resultant solution was
stirred for one hour to remove a Boc group and an Ot-Bu group.
Then, the sample was washed by repeating re-precipitation in ether,
and was dried under reduced pressure, and white solid
Val-Lys-polyrotaxane conjugate (113 mg) was obtained. Other six
types of Val-Lys-polyrotaxane conjugates (VK-PRX: chemical formula
1) and two types of Val-Lys-.alpha.-CD (VK-.alpha.-CD) were
synthesized by using each reagent with the amount shown in Table 4
and Table 5, by a method similar to the above-mentioned method.
4TABLE 4 VK- OH group in PRX CDI-PRX Val-Lys DIPEA AMEt DMSO
Synthesized No. (mmol) (mmol) (mmol) (mmol) (ml) amount (mg) 1 3.17
3.21 3.5 -- 2 198 2 3.02 6.31 7.2 -- 2 195 3 3.20 0.69 6.9 33 2 93
4 3.29 6.55 3.5 33 2 95 5 3.29 6.54 5.0 33 2 80 6 3.07 6.21 7.5 33
2 37 7 1.76 3.25 3.5 33 2 113
[0052]
5TABLE 5 OH group in VK-.alpha.- CDI-PRX Val-Lys DIPEA AMEt DMSO
Synthesized CD No. (mmol) (mmol) (mmol) (mmol) (ml) amount (mg) 1
5.56 0.62 2.68 -- 5 297 2 5.56 0.62 2.68 5.6 5 338
[0053] (Chemical Formula 1) 1
Example 4
[0054] [Characterization of Val-Lys-polyrotaxane Conjugate]
[0055] 4-1 (Calculation of the number of penetrated .alpha.-CDs and
introduced hydroxyehylcarbamoyl groups in Val-Lys-polyrotaxane
conjugates)
[0056] The number of penetrated .alpha.-CDs in the seven types of
VK-PRX mentioned above (.alpha.-CD/PRX) was calculated from the
integral values of .sup.1HNMR spectrum. The results are shown in
Table 6.
6TABLE 6 VK-PRX No. M.sub.w .alpha.-CD/PRX Val-Lys/PRX AMEt/PRX 1
25,300 21 1 2 19,900 14 7 3 30,560 22 1 71 4 34,600 25 2 86 5
24,100 16 2 57 6 18,240 9 11 36 7 43,200 21 46 98
[0057] 4-2 (Quantitatation of the number of introduced Val-Lys in
VK-PRX and VK-.alpha.-CD by amino acid analysis)
[0058] A small amount (1.about.2 mg) of each of the seven types of
VK-PRX and the two types of VK-.alpha.-CD obtained in Example 3
were dissolved in 6N HCl, and N.sub.2 substitution was conducted.
Next, thermolysis was performed at 110.degree. C. for about 22
hours. After removing HCl completely, the resultant solution was
diluted with 0.02 N HCl (2.about.4 ml), and made to be a sample.
The sample thus made was quantitated with an amino-acid analyzer
(Hitachi amino-acid analyzer; L-8500A). Based on the composition of
a Phe residue and a Val residue on both ends of VK-PRX or
VK-.alpha.-CD obtained by the amino-acid analysis, the number of
Val-Lys in one molecule of VK-PRX or VK-.alpha.-CD (Val-Lys/VK-PRX
or Val-Lys/VK-.alpha.-CD) was calculated with the following two
formulas (Table 6 and Table 7). The two formulas were reached as
follows. In addition, with regard to AMEt/PRX in Table 6 and
AMEt/.alpha.-CD in Table 7, the number of aminoethanol (AMET)
molecules introduced into VK-PRX or VK-.alpha.-CD were calculated
based on the ratio of the integral values of methylene peak from
aminoethanol observed on the proton nuclear magnetic resonance
(.sup.1H-NMR) spectrum to the methine peak at an anomeric position
in .alpha.-CD.
7 TABLE 7 VK-.alpha.-CD No. Mw Val-Lys/.alpha.-CD AMEt/.alpha.-CD 1
1,220 1 2 1,400 1 3
[0059] As one molecule of Val-Lys-AMEt-RX has two molecules of Phe
residues, when the number of moles of Phe residues in the sample is
n.sub.phe, the number of moles of Val-Lys polyrotaxane conjugate
(n.sub.RX) present in the sample to be measured is shown by the
formula (1).
[0060] (Mathematical Formula 1) 1 n RX = n Phe 2 ( 1 )
[0061] Further, when the number of moles of Val residues is
n.sub.val, and the number of moles of Val-Lys polyrotaxane
conjugate is n.sub.RX, the number of introduced Val-Lys in one
molecule of Val-Lys polyrotaxane conjugate (N.sub.Val-Lys) is shown
by the formula (2).
[0062] (Mathematical Formula 2) 2 N Val - Lys = n Val n RX ( 2
)
Example 5
[0063] [Examination of Substrate Recognition Property to a
Polymeric PEPT 1 Inhibitor Using HeLa-hPEPT 1 Cells]
[0064] In order to examine the recognition property of VK-PRX and
its component, VK-.alpha.-CD, both obtained in Example 4, to PEPT
1, the inihibitory effect to the uptake of [.sup.3H] Gly-Sar by
using HeLa cells stably expressing hPEPT 1 (HeLa-hPEPT 1) was
examined. HeLa-hPEPT 1 cells or HeLa-pcDNA (Mock) cells prepared by
the method described previously (Int. J. Cancer. 88, 274-80, 2000)
were placed in a multidish (Nunc) at 10.sup.6 cells/well and
cultured in an incubator (Hirasawa) at 37.degree. C. and 5%
CO.sub.2 for four days. As a broth, DMEM (Dulbecco's modified
Eagle's medium; Gibco Laboratories) containing 10% FCS (Gibco
Laboratories), 2 mM L-glutamine, and 1 mg/ml G418 was used. After
culture, the broth was aspirated and each cell was washed three
times with 1 ml of Hanks' balanced salt solution (HBSS; 0.952 mM
CaCl.sub.2, 5.36 mM KCl, 0.441 mM KH.sub.2PO.sub.4, 0.812 mM
MgSO.sub.4, 136.7 mM NaCl, 0.385 mM Na.sub.2HPO.sub.4, 25 mM
D-glucose, 10 mM MES: pH 6.0) at 37.degree. C., then preincubated
for five minutes. Subsequently, 250 .mu.l of HBSS containing each
inhibitor with the concentration shown in FIG. 4 and Table 8, and
[.sup.3H(G)] Gly-Sar (476 nM) was added and an uptake reaction was
initiated at 37.degree. C. The inhibitors, VK-PRX (No. 1.about.6),
were prepared such that the highest concentration of dissolved
VK-PRX would be 500 .mu.M or lower, and were filtrated through a
filter, and then used for the uptake reaction. At the beginning of
the reaction, the reaction solution was taken from each dish and
placed into 10 .mu.l mini vial. Then, 4 ml of liquid scintillation
cocktail (Clear-sol I, Nacalai tesque) was added to the solution,
and radioactivity in the reaction solution was measured by a liquid
scintillation counter (LSC-5100, Aloka Co. Ltd.). Two minutes after
the beginning of the reaction, the reaction solution in each dish
was removed by aspiration, and the reaction was stopped by washing
the cells three times with 1 ml of ice-cooled HBSS (HEPES: pH 7.4).
Subsequently, 250 .mu.l of 5 N NaOH was added to each dish to
solubilize the cells (for two hours or longer), and 250 .mu.l of 5
N HCl was added for neutralization, then the whole amount was
poured into mini vial, and 4 ml of liquid scintillation cocktail
(Clear-sol I, Nacalai tesque) was added, and radioactivity taken in
the cells was measured.
[0065] Further, in order to measure the amount of cellular proteins
after the culture mentioned above, the cultured cells were
solubilized, Bio-Rad Protein Assay reagent (Bio-Rad Co.) was added,
and the absorbance at 595 nm was measured. As a standard, BSA
(bovine serum albumin) was used. Based on the results, the uptake
amount of [.sup.3H] Gly-Sar into the cells [cell/medium ratio
(.mu.l/mg.multidot.protein)] was calculated according to the
formula (3). The results are shown in FIG. 4 and Table 8. The value
of the control in FIG. 4 is the result of the uptake reaction
conducted in the absence of the inhibitor. These results have
indicated that VK-PRXs (No. 1, 2, 4, 6) reduce the uptake of
[.sup.3H] Gly-Sar significantly. It has been shown that the uptake
of [.sup.3H] Gly-Sar is also inhibited concentration-dependently in
the case where VK-PRX (No. 7) and VK-.alpha.-CD are used. However,
as to .alpha.-CD to which Val-Lys was not bound, significant
inhibitory effect was not observed.
[0066] (Mathematical Formula 3) 3 cell / medium ratio ( L / mg
protein ) = radioactivity taken in the cells ( dpm / well )
radioactivity concentration in chemicals ( dpm / mL ) .times. the
amount of proteins ( mg protein / well ) ( 3 )
8 TABLE 8 Concentration [.sup.3H] Gly-Sar uptake Inhibitor (mM) %
of control Gly-Sar 10 12.93 .+-. 2.45 Val-Lys 3 21.13 .+-. 1.80
VK-.alpha.-CD (No. 1) 3 37.05 .+-. 1.52 VK-.alpha.-CD (No. 2) 3
77.09 .+-. 5.67 .alpha.-CD 3 125.87 .+-. 23.34 VK-PRX (No. 1)
<0.5 37.95 .+-. 1.82 VK-PRX (No. 2) <0.5 76.68 .+-. 4.62
VK-PRX (No. 3) <0.5 131.12 .+-. 3.75 VK-PRX (No. 4) <0.5
67.32 .+-. 8.57 VK-PRX (No. 5) <0.5 94.04 .+-. 4.21 VK-PRX (No.
6) <0.5 77.20 .+-. 3.12 VK-PRX (No. 7) 1 52.93 .+-. 3.69 VK-PRX
(No. 7) 0.5 76.08 .+-. 5.82 VK-PRX (No. 7) 0.3 83.95 .+-. 10.78
VK-PRX (No. 7) 0.1 104.43 .+-. 8.70 Cefadroxil 10 27.69 .+-. 1.44
Cephalexin 10 70.12 .+-. 1.45 Gly 10 97.71 .+-. 7.10
Example 6
[0067] [The Effect of Preincubation of a Polymeric PEPT 1
Inhibitor]
[0068] In order to examine the change of recognition property to
PEPT 1 in polyrotaxanation of VK-.alpha.-CD, the influence of
preincubation of the cells with VK-PRX (No. 7) and VK-.alpha.-CD
(No. 2) on [.sup.3H] Gly-Sar uptake was examined by using HeLa
cells stably expressing hPEPT 1 (HeLa-hPEPT 1). In the presence or
absence of each inhibitor with concentrations shown in FIG. 5,
preincubation was conducted for 30 minutes (open columns), and
after the reaction solution was removed, the inhibitory effect on
[.sup.3H] Gly-Sar uptake was measured in a manner similar to that
of Example 5 except that the uptake reaction was conducted in HBSS
containing the same concentration of the inhibitor and [.sup.3H]
Gly-Sar (476 nM) (closed columns). The results are shown in FIG. 5.
In the figure, "Mock" indicates the uptake by HeLa-pcDNA 3 cells.
Thus, the preincubation with VK-PRX (No. 7) or VK-.alpha.-CD (No.
2) affected the uptake of [.sup.3H] Gly-Sar. With regard to VK-PRX
(No. 7), significant decrease in the uptake was observed while
significant increase in the uptake was observed with regard to
VK-.alpha.-CD (No. 2). In addition, VK-PRX (No. 7), which is a
supramolecular form of VK-.alpha.-CD (No. 2), showed stronger
inhibitory effect, however, no inhibitory effect of .alpha.-CD to
which Val-Lys was not bound was observed.
Example 7
[0069] [Evaluation of Absorption Inhibitory Effect of VK-PRX by
Changes in Pharmacokinetics of Cefadroxil (CDX)]
[0070] Recently, it is reported that T-1095, a glucose transporter
inhibitor in the kidney, ameliorates hyperglycemic conditions in
STZ rats (Streptozotocin-induced diabetic rats)(Metabolism, 49,
990-5, 2000). This report suggests that clinical conditions could
be ameliorated by suppressing the transporter function and
controlling the transportation of bioactive substances, and there
is an expectation as a drug target. Further, it has been reported
that bioavailability of cefadroxil [CDX: (chemical formula 2)], a
.beta.-lactam antibiotic which is a substrate of PEPT 1 in human,
is decreased by the coadministration of cephalexin [CEX: (chemical
formula 3)] which is a .beta.-lactam antibiotic (Eur. J. Clin.
Pharmacol. 41, 179-83, 1991). This report indicates that absorption
of CDX through the digestive tract via PEPT 1 was suppressed by
CEX, which is also a substrate of PEPT 1. Therefore, the inhibitory
effect of a PEPT 1 inhibitor on CDX absorption was examined by
using rats.
[0071] (Chemical Formula 2) 2
[0072] (Chemical Formula 3) 3
[0073] SD (Sprague-Dawley) rats (male; Japan SLC, Inc.) of
7.about.8 weeks of age were anesthetized by intraperitoneal
administration of 50 mg/kg of Nembutal (Dainabbot, Inc.), and after
fixing their backs, jugular veins and femoral veins (only when
instant intravenous injection was performed) were cannulated
[silicon tube, inner diameter.times.outer diameter (0.5.times.1)],
and the tip end of the cannula was inserted subcutaneously and
passed through from the back of the neck, then the cut was stitched
up. Subsequently, the animals were starved overnight (about 18
hours) and each pharmaceutical was administered orally via oral
sonde, or administered via femoral vein. After administering each
pharmaceutical, 400 .mu.l of the blood was collected from the
cannulation tube over time, and CDX concentration in the plasma was
measured by the method mentioned below, then each parameter was
calculated by the formula (4) or the formula (5), the formula (6),
the formula (7), and the formula (8), and analyzed according to the
compartment model analysis of WinNonlin (Scientific Consulting
Inc.) (Edited by Akira Tsuji, "Comprehensible Biopharmaceutics",
Hirokawa Shoten, 178-188, 1996).
[0074] 400 .mu.l of the collected blood mentioned above was
substituted with the same amount of saline, and the blood was
dispensed into 1.5 ml microtube, and centrifuged (12000 rpm, 5
minutes, 4.degree. C.) to extract the plasma. 150 .mu.l of the
plasma was transferred to 1.5 ml microtube, and added with the same
amount of acetonitrile for deprotein treatment. After
centrifugation (12000 rpm, 5 minutes, 4.degree. C.), 250 .mu.l of
supernatant was transferred to 1.5 ml microtube, and evaporated to
dryness, then reconstructed according to the HPLC condition
mentioned below. As a column, TSKgel ODS-80Ts (Toyo Soda) was used,
and as a pump, an ultraviolet-visible light detector, an
intelligent autosampler, and a column oven, 880-PU, 875-UV,
AS-1555-10, and Co-1565 (all from JASCO Corporation) were used
respectively, and as an integrator, Chromatopac C-R3A (Shimadzu
Corporation) was used. At the column temperature 35.degree. C., 7%
of acetonitrile (containing 0.1 M of acetic acid buffer (pH 3.0)
and 0.01 M of 1-pentasulfonic acid sodium) was used as a mobile
phase for separating elution at flow rate 0.9 ml/min. The detection
was conducted at wavelength 240 nm, and CDX concentration in the
plasma was determined.
[0075] (Mathematical Formula 4) 4 C = ka F D { exp ( - ke t ) - exp
( - ka t ) } Vd ( ka - ke ) ( 4 )
[0076] In the formula, C, F, D, ka, ke, Vd and t represent CDX
concentration in the plasma, absorptance, dosage, absorption rate
constant, elimination rate constant, distribution volume, and time
interval between the drug administration and blood collection,
respectively.
[0077] (Mathematical Formula 5)
C=A.multidot.exp(-.alpha..multidot.t)+B.multidot.exp(-.beta..multidot.t)
(5)
[0078] With the proviso that A=D
(k.sub.21-.alpha.)/V.sub.1(.beta.-.alpha.- )
[0079] B=D (k.sub.21-.beta.)/V.sub.1(.alpha.-.beta.)
[0080] In the formula, .alpha., .beta. and V.sub.1 represent
gradient of distribution phase, gradient of elimination phase, and
distribution volume of central compartment, respectively.
[0081] (Mathematical Formula 6)
AUC.sub.0-.infin.=AUC.sub.0-6+C.sub.6/ke (6)
[0082] In the formula, AUC represents area under the plasma
concentration-time curve, C.sub.6 represents concentration of a
drug in the plasma 6 hours after the administration,
respectively.
[0083] (Mathematical Formula 7)
CL=Dose/AUC.sub.0-.infin. (7)
[0084] In the formula, CL represents total clearance, Dose
represents the dose of the drug, respectively.
[0085] (Mathematical Formula 8)
Vd=CL/ke (8)
[0086] b 7-1 (The Inhibitory Effect of CEX on CDX Transportation
Via PEPT 1 Using Rats)
[0087] It has been reported that bioavailability of CDX, which is a
substrate of PEPT 1, is decreased by the coadministration of CEX,
which is a .beta.-lactam antibiotic in human (Eur. J. Clin.
Pharmacol., 41, 179-83, 1991). In order to elucidate the inhibitory
effect of VK-PRX on PEPT 1, 2.5 mg/kg CDX (.smallcircle.), 5 mg/kg
CDX (.circle-solid.), or 2.5 mg/kg CDX and 45 mg/kg CEX
(.tangle-soliddn.) as pharmaceuticals were orally administered to
the rats mentioned above, and it was examined whether CEX inhibited
the absorption of CDX in rats by the method described in Example 7.
With regard to 2.5 mg/kg CDX and 45 mg/kg CEX (.tangle-soliddn.),
CEX was orally administered to the rats 30 minutes before the
administration of CDX. Each pharmacokinetic parameter of the
above-mentioned rats was calculated based on the change in the CDX
concentration in the blood, with the formula (4), the formula (6),
the formula (7), and the formula (8), and evaluated. The results
are shown in FIG. 6 and Table 9. Values shown in the figure and the
table are the mean.+-.S.E.M. of three to four independent
experiments. The results of oral administration of CDX at 2.5 mg/kg
or 5 mg/kg have shown saturation phenomenon in AUC.sub.0-.infin.
and Cmax. In addition, when CDX was administered at 2.5 mg/kg, the
sufficient absorptance, 86%, was obtained. It has been shown that
CDX is appropriate as a marker compound which evaluates absorption
activity via PEPT 1, taking into account that CDX is stable in a
living organism. Further, in the case where CDX (2.5 mg/kg) was
administered after CEX (45 mg/kg) was preadministered, area under
the plasma concentration-time curve (AUC.sub.0-.infin.) was
decreased by about 30%, absorption rate constant (ka) and maximum
plasma concentration (Cmax) were significantly decreased from 2.42
to 1.53 hr.sup.-1 and 0.8 to 0.5 .mu.g/ml, respectively, in
comparison to the case where CDX alone was administered. The above
results have reproduced the report, which deals with the cases of
human, in rats.
9 TABLE 9 Dose (mg/kg) 2.5 5 2.5 + 45 ka (hr.sup.-1) 2.42 .+-. 0.08
2.09 .+-. 0.23 1.53 .+-. 0.27* ke (hr.sup.-1) 0.33 .+-. 0.02 0.31
.+-. 0.03 0.37 .+-. 0.03 Tmax (hr) 0.95 .+-. 0.03 1.09 .+-. 0.08
1.25 .+-. 0.13 Cmax (.mu.g/mL) 0.80 .+-. 0.03 0.56 .+-. 0.07 0.50
.+-. 0.04 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 198 .+-. 4.82
162 .+-. 13.3 144 .+-. 5.21
[0088] 7-2 (The Change in Absorption Inhibitory Effect by VK-PRX
Type)
[0089] Next, 5 mg/kg CDX alone (.smallcircle.), or 10 mg/kg VK-PRK
[VK-PRK suspended in 0.1% of sodium polyacrylate (PANA) in saline]
and 5 mg/kg CDX (.circle-solid.) were used as pharmaceuticals and
orally administered to the rats mentioned above, and it was
examined whether VK-PRX inhibited the absorption of CDX in rats by
the method described in Example 7. The above-mentioned VK-PRK was
administered to the rats 30 minutes before the administration of 5
mg/kg CDX (.circle-solid.). Then each pharmacokinetic parameter was
calculated based on the change in the CDX concentration in the
blood, with the formula (4), the formula (6), the formula (7), and
the formula (8), and evaluated. The results obtained with VK-PRK
(No. 2) are shown in FIG. 7 and Table 10, and the results obtained
with VK-PRK (No. 7) are shown in FIG. 8 and Table 11. Values shown
in the figures are the mean.+-.S.E.M. of two to four independent
experiments, and values shown in the tables are the mean.+-.S.E.M.
of three independent experiments. VK-PRK (No. 2) or VK-PRK (No. 7)
was coadministered with CDX, but there was no significant
difference observed in AUC.sub.0-.infin. in comparison to the case
where CDX alone was administered. Further, there was no significant
difference observed with regard to other parameters, either.
Judging from these results, it is presumed that the dose of CDX (5
mg/kg) mentioned above was a condition wherein the effect of VK-PRX
(No. 2) was difficult to be detected because the absorption through
the digestive tract had already been saturated.
10 TABLE 10 Parameter CDX CDX + VK-RX (2) ka (hr.sup.-1) 1.50 .+-.
0.24 3.21 .+-. 0.18 ke (hr.sup.-1) 0.54 .+-. 0.06 0.49 .+-. 0.10
Tmax (hr) 1.13 .+-. 0.11 0.70 .+-. 0.02 Cmax (.mu.g/m) 1.22 .+-.
0.08 1.17 .+-. 0.28 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 272
.+-. 9.27 244 .+-. 93.4
[0090]
11 TABLE 11 Parameter CDX CDX + VK-RX (7) ka (hr.sup.-1) 1.50 .+-.
0.24 3.33 .+-. 0.00 ke (hr.sup.-1) 0.54 .+-. 0.06 0.63 .+-. 0.25
Tmax (hr) 1.13 .+-. 0.11 0.64 .+-. 0.10 Cmax (.mu.g/mL) 1.22 .+-.
0.08 1.04 .+-. 0.11 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 272
.+-. 9.27 220 .+-. 20.2
[0091] 7-3 (The Dose of VK-PRX and its Effect)
[0092] As in the method described in Example 7, 5 mg/kg CDX alone
(.smallcircle.), 10 mg/kg CEX and 5 mg/kg CDX (.tangle-soliddn.),
or 5.7 mg/kg VK-PRK (No. 2) and 5 mg/kg CDX (.circle-solid.) were
orally administered to rats simultaneously, and each
pharmacokinetic parameter was calculated and evaluated. The results
are shown in FIG. 9 and Table 12. Values shown in the figure and
the table are the mean.+-.S.E.M. of two to four independent
experiments. As in the case where 10 mg/kg VK-PRX (No. 2) was
coadministered with CDX (FIG. 7 and Table 10), in the case where
5.7 mg/kg VK-PRX (No. 2) was coadministered with CDX, there was no
significant difference observed in AUC.sub.0-.infin. in comparison
to the case where CDX alone was administered. Further, there was no
significant difference observed with regard to other parameters,
either.
12TABLE 12 Parameter CDX CDX + CEX CDX + VK-PRX (2) ka (hr.sup.-1)
1.50 .+-. 0.24 2.41 .+-. 0.49 1.37 .+-. 0.73 ke (hr.sup.-1) 0.54
.+-. 0.06 0.68 .+-. 0.19 0.49 .+-. 0.13 Tmax (hr) 1.13 .+-. 0.11
0.79 .+-. 0.07 1.32 .+-. 0.29 Cmax (.mu.g/mL) 1.22 .+-. 0.08 1.29
.+-. 0.21 1.32 .+-. 0.08 AUC.sub.0-.infin. 272 .+-. 9.27 247 .+-.
23.6 337 .+-. 29.9 (.mu.g .multidot. min/mL)
[0093] 7-4 (The Influence of a Suspending Agent on VK-PRX
Effect)
[0094] Next, in order to examine the difference in the effect of
VK-PRX between the case where each pharmaceutical was suspended in
saline containing 0.1% of sodium polyacrylate (PANA) which is a
suspending agent (FIG. 10), and the case where each pharmaceutical
was not suspended (FIG. 11), 10 mg/kg VK-PRK (No. 7) and 2.5 mg/kg
CDX (.circle-solid.), or 2.5 mg/kg CDX alone were orally
administered, and each pharmacokinetic parameter in each rat was
calculated according to the method described in Example 7 and
evaluated. VK-PRK was orally administered 30 minutes before CDX
administration. The results of the case where each pharmaceutical
was suspended in PANA are shown in FIG. 10 and Table 13, and the
results of the case where each pharmaceutical was not suspended in
PANA are shown in FIG. 11 and Table 14. Values shown in FIG. 10,
FIG. 11 and Table 14 are the mean.+-.S.E.M. of four independent
experiments, and values shown in Table 13 are the mean.+-.S.E.M. of
three independent experiments. These results have indicated that in
the case where VK-PRK (No. 7) was suspended in a suspending agent
and coadministered with CDX, there was no significant difference
observed in AUC.sub.0-.infin. in comparison to the case where CDX
alone was administered, and that there was no significant
difference observed with regard to other parameters, either. On the
other hand, it has been found that AUC.sub.0-.infin. significantly
decreases when VK-PRK is administered under the same condition
excluding the suspension in PANA. Further, by coadministration of
VK-PRK, ka and Cmax were significantly decreased from 2.42 to 1.75
hr.sup.-1 and 0.8 to 0.64 .mu.g/ml, respectively, and Tmax was
significantly prolonged from 0.95 to 1.18 hr. However, no
significant difference was observed in the elimination rate
constant (ke).
13 TABLE 13 Parameter CDX CDX + VK-RX (7) ka (hr.sup.-1) 2.12 .+-.
0.45 1.88 .+-. 0.03 ke (hr.sup.-1) 0.37 .+-. 0.02 0.33 .+-. 0.02
Tmax (hr) 1.04 .+-. 0.12 1.13 .+-. 0.04 Cmax (.mu.g/mL) 0.76 .+-.
0.04 0.69 .+-. 0.02 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 186
.+-. 9.15 188 .+-. 7.42
[0095]
14 TABLE 14 Parameter CDX CDX + VK-RX (7) ka (hr.sup.-1) 2.42 .+-.
0.08 1.75 .+-. 0.19 ke (hr.sup.-1) 0.33 .+-. 0.02 0.34 .+-. 0.03
Tmax (hr) 0.95 .+-. 0.03 1.18 .+-. 0.05 Cmax (.mu.g/mL) 0.80 .+-.
0.03 0.64 .+-. 0.05 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 198
.+-. 4.82 175 .+-. 6.26
[0096] 7-5 (The Effectiveness of Preadministration of VK-PRX)
[0097] The effectiveness of preadministration of VK-PRX was also
examined. Each pharmacokinetic parameter, in the case where 2.5
mg/kg CDX and 10 mg/kg VK-PRX (No. 7) were orally coadministered
(.circle-solid.), or 2.5 mg/kg CDX alone were orally administered
(.smallcircle.), was calculated in the same manner as the method
described in Example 7 and evaluated. The results are shown in FIG.
12 and Table 15. Values shown in the figure are the mean.+-.S.E.M.
of three independent experiments, and values shown in the table are
the mean.+-.S.E.M. of four independent experiments. As the results,
in the case where VK-PRX (No. 7) was coadministered with CDX, there
was no significant difference observed in AUC.sub.0-.infin. in
comparison to the case where CDX alone was administered. However,
in the case where VK-PRX (No. 7) was preadministered and CDX was
administered 30 minutes later (FIG. 11 and Table 14), significant
decrease of AUC.sub.0-.infin. was observed, and significant
decrease of ka was observed regardless of whether preadministration
was conducted or not.
15 TABLE 15 Parameter CDX CDX + VK-RX (7) ka (hr.sup.-1) 2.42 .+-.
0.08 1.29 .+-. 0.28 ke (hr.sup.-1) 0.33 .+-. 0.02 0.57 .+-. 0.13
Tmax (h) 0.95 .+-. 0.03 1.18 .+-. 0.08 Cmax (.mu.g/mL) 0.80 .+-.
0.03 0.78 .+-. 0.08 AUC.sub.0-.infin. (.mu.g .multidot. min/mL) 198
.+-. 4.82 175 .+-. 9.98
[0098] Example 8
[0099] [The Influence after VK-PRX Intravenous Injection on
Pharmacokinetics of CDX]
[0100] Since VK-PRX is a polymer compound, it is not presumed to be
absorbed through the digestive tract. However, it is also known
that orally administered CEX prompts the excretion of CDX, and
therefore, it is not necessarily explicable if the effect of VK-PRX
that decreases AUC.sub.0-.infin. of CDX means the decrease of
absorbency only. On the other hand, CDX is known to show
nonlinearity in the reabsorption through the kidney (Drug Metab.
Dispos. 21, 215-7, 1993, Drug Metab. Dispos. 22, 447-50, 1994). In
addition, oligopeptide transporters are involved in the
reabsorption. Therefore, in order to compare the influence on CDX
clearance (CL: renal excretion) in rats instantly and intravenously
injected with 2.5 mg/kg CDX, and simultaneously administered with
10 mg/kg VK-PRX (No. 7) [VK-PRK dissolved in saline] orally
(.smallcircle.), to that in rats instantly and intravenously
injected with 2.5 mg/kg CDX (.circle-solid.), each pharmacokinetic
parameter was calculated according to the method described in
Example 7 and evaluated (FIG. 13 and Table 16). Values shown in the
figure and the table are the mean.+-.S.E.M. of three independent
experiments. As the results, the administration of VK-PRX did not
affect the change in the CDX concentration in the plasma,
indicating that the change of AUC.sub.0-.infin. mentioned above was
caused not by the influence on renal excretion/reabsorption
process, but by absorption through the digestive tract. Further, it
has been already revealed that physical stability of a peptide
carried by PRX increases (Pharm. Res. 16, 1331-1343, 1999), and the
obtained results support that VK-PRX is a compound which is not
absorbed or is hard to be absorbed through the digestive tract.
These results have revealed that a polymeric PEPT 1 inhibitor as a
nonabsorbent compound inhibits the PEPT 1-mediated absorption. This
result leads to the suppression of PEPT 1-mediated protein
absorption.
16 TABLE 16 Parameter CDX CDX + VK-RX (7) AUC.sub.0-.infin. (.mu.g
.multidot. min/mL) 229 .+-. 8.41 222 .+-. 5.09 Vdss (mL) 162 .+-.
8.99 134 .+-. 10.3 CLtot (mL/min) 11.0 .+-. 0.41 11.3 .+-. 0.26 ke
(hr.sup.-1) 4.10 .+-. 0.28 5.12 .+-. 0.46
INDUSTRIAL APPLICABILITY
[0101] As the tissue-specific transporter inhibitor of the present
invention is not absorbed or is hard to be absorbed through the
small intestine, it can prevent deterioration in the QOL, which is
caused by diet therapy, of patients who suffer from tissue
dysfunction diseases or renal failure by specifically decreasing
the absorption of nutrients through the small intestine. In
addition, the tissue-specific transporter inhibitor is useful for
preventive medicine that prevents the onset of tissue diseases such
as renal diseases and conservative treatments that prevent the
progress of renal failure into dialysis.
[0102] The invention will now be further described by the following
numbered paragraphs:
[0103] 1. A tissue-specific transporter function inhibitor which
has both a ligand structure recognized by a tissue-specific
transporter and a polymeric molecular structure incapable of
passing through a membrane tissue.
[0104] 2. The tissue-specific transporter function inhibitor
according to paragraph 1, wherein the polymeric molecular structure
incapable of passing through a membrane tissue is a supramolecular
structure.
[0105] 3. The tissue-specific transporter function inhibitor
according to paragraph 2, wherein the supramolecular structure is a
rotaxane compound in which a number of circular molecules are
penetrated by linear molecules, and both ends of the linear
molecules are capped by bulky substituents.
[0106] 4. The tissue-specific transporter function inhibitor
according to paragraph 3, wherein the circular molecules are
cyclodextrins.
[0107] 5. The tissue-specific transporter function inhibitor
according to paragraph 3 or 4, wherein the linear molecules are
polyethyleneglycols.
[0108] 6. The tissue-specific transporter function inhibitor
according to any one of paragraphs 3 to 5, wherein the bulky
substituents are N-benzyloxycarbonyl-L-phenylalanines.
[0109] 7. The tissue-specific transporter function inhibitor
according to paragraph 1, wherein the polymeric molecular structure
incapable of passing through a membrane tissue is an
.alpha.-cyclodextrin structure.
[0110] 8. The tissue-specific transporter function inhibitor
according to any one of paragraphs 1 to 7, wherein the ligand
recognized by a tissue-specific transporter is an organic anionic
substance, an organic cationic substance, or a peptidergic
substance.
[0111] 9. The tissue-specific transporter function inhibitor
according to any one of paragraphs 1 to 8, wherein the
tissue-specific transporter is a small intestine-specific
transporter.
[0112] 10. The tissue-specific transporter function inhibitor
according to paragraph 9, wherein the small intestine-specific
transporter is an oligopeptide transporter 1 (PEPT 1).
[0113] 11. The tissue-specific transporter function inhibitor
according to paragraph 10, wherein a peptidergic substance
recognized by the oligopeptide transporter 1 (PEPT 1) is
valyl-lysine (Val-Lys).
[0114] 12. A therapeutic drug for tissue dysfunction diseases which
contains the tissue-specific transporter function inhibitor
according to any one of paragraphs 1 to 11 as an active
ingredient.
[0115] 13. A therapeutic drug for suppressing the progress of
chronic renal failure which contains the tissue-specific
transporter function inhibitor according to any one of paragraphs 1
to 11 as an active ingredient, wherein the inhibitor is a protein
absorption inhibitor.
* * * * *